Rapid and sensitive method for determination of catecholamines in human plasma

Abstract

Various methods1-4 using trihydroxyindol reaction for the measurement of catecholamines in human plasma have been designed by workers hitherto. However, in these methods1, 3, 4, isolation of the catecholamines with an adsorbent presents considerable difficulties. Thus, in spite of the availability of these methods1, 3, 4 reproduction by other laboratories are difficult. The main objective of the present work was to improve the method for simultaneous determination of epinephrine (Ep.) and norepinephrine (NE.) in human plasma. A preliminary experiment was set up in order to run a comparison against these methods with respect to the recovery, accuracy and rapidity. Of these methods PRICE's method3 showed satisfactory recovery and rapidity. Thus several improvements of PRICE's methods3 were made: 1) Purification of acid-washed aluminum oxide (Merck acid washed #71695) was made. 2) A large amount of EDTA disodium was used in several steps. 3) The timing procedure becomes essential due to the fact that fluorophor of both Ep. and to a smaller degree NE. decays at a considerable rate. Thus, a means with sufficiently high accuracy and rapidity was obtained. Material and Method A preparation of acid-washed aluminum oxide (for chromatographic use). a) 300-500 g of alumina (Merck-71695 acid washed) was boiled using a reflux condenser with frequent shaking in 1 L 2N HCl for 1 hour. The slurry was allowed to cool to room temperature. This was transferred to a 1 L chromato-graphic tube with a fritter disc (pyrex #38450). and was cycled with deionized water until the pH of the supernatant rose to 5.0 after which a stirring of the column was followed by cleaning of the supernatant for 2 minutes. This rapid cleaning indicates that most of the fine grain alumina is removed. Next collect the alumina in a suction funnel with filter paper, allow to dry overnight on large sheet of filter paper (Whatman No.3) at room temperature. Then heat in an oven at 200°C for two hours, place in desiccator. b) All solutions of mineral acids, bases, and buffers were prepared from reagent grade chemicals with water previously distilled and passed through a commercial deionizer. Water purified in this manner is used throughout the procedure.